Patent Application
20090203586
The present invention relates, inter alia, to a method of ameliorating an inflammatory skin condition.
Inflammatory skin conditions are known to be associated with chemokines and cytokines, and in particular the activities of pro-inflammatory cytokines such as IL-1α, IL-1β and tumour necrosis factor a (TNF-α). These same cytokines are known also to play pivotal roles in the initiation of skin immune responses, and in fact provide mandatory signals for the migration of epidermal Langerhans cells (LC) from the skin. The movement of LC from the skin, and their subsequent accumulation in skin-draining lymph nodes provides a mechanism for the transport of antigen to the sites (regional lymph nodes) where immune responses are induced.
Our understanding that the migration of LC from the epidermis is dependent upon the provision of signals by IL-1α, IL-1β and TNF-α provides an experimental system for investigating the availability and functional activity of these cytokines in skin tissues. Experience has shown that factors that are known to inhibit the availability or function of IL-1α, IL-1β or TNF-α are associated with a significant inhibition of induced LC migration.
In addition to being required for the stimulation of LC mobilisation, IL-1β is known to cause skin inflammation and has been implicated, directly or indirectly, in the pathogenesis of several cutaneous inflammatory disorders. IL-1β is synthesised as an inactive intracellular precursor protein, which is cleaved and secreted to yield mature carboxy-terminal fragments that are biologically active and exert their effect by binding to specific cell surface receptors found on almost all cell types and triggering a range of responses.
The present invention is based on the surprising discovery that certain molecules are able, when applied topically to the skin, to inhibit the production and/or availability of bioactive IL-1α and/or IL-1β. As such these molecules are suitable, inter alia, for the treatment of inflammatory skin conditions where IL-1α and/or IL-1β are implicated in the pathogenesis. Suitable molecules include thioredoxin (TRX), a 12-kDa protein with a Cys-Gly-Pro-Cys active site, and additionally “redox-inactive” TRX molecules, wherein the cysteines at the active site are replaced by amino acids other than cysteine. Whilst it has been shown that these molecules are likely to exert their therapeutic effect by inhibiting the production or activity of IL-1α or IL-1β—it is also possible that they exert an associated or additional beneficial effect by stimulating the production of anti-inflammatory cytokine(s), such as interleukin-10 (IL-10).
According to the present invention there is provided a polypeptide capable of ameliorating an inflammatory skin condition wherein said polypeptide is a modified thioredoxin, the modification comprising:
Preferably, the modification consists of independently substituting both Cys1 and Cys2 with an amino acid other than Ser. The modification of the active site renders the active site redox-inactive and, surprisingly, it has been found that such redox-inactive molecules are capable of ameliorating an inflammatory skin condition.
The present invention further provides a modified thioredoxin wherein if the unmodified thioredoxin contains one or more cysteines in addition to Cys1 and Cys2, then the modification further comprises substituting and/or deleting one or more of the additional cysteines.
Both Cys1 and Cys2 may be independently substituted. For example, one embodiment of the polypeptide of the present invention could comprise Ser-Gly-Pro-Ala, another Ala-Gly-Pro-Ser. In a preferred embodiment of the invention Cys1 and Cys2 are both substituted by Ala to give Ala-Gly-Pro-Ala. More preferred is a polypeptide wherein the unmodified TRX is human TRX, and more preferred still is the polypeptide selected from the group consisting of SEQ ID NO. 3, SEQ ID NO. 9 and SEQ ID NO. 10.
A further embodiment of the present invention is a DNA sequence that encodes a polypeptide of the present invention. The exact nature of the DNA sequence would, of course, depend on the specific nature of the polypeptide and the intended use of the DNA sequence. For example, codon-optimisation of the DNA sequence may be required for expression of the DNA sequence in a recombinant expression system (an example of a codon-optimised sequence is provided as SEQ ID NO. 6). The techniques required to provide such DNA sequences are well within the knowledge of the skilled man. A preferred DNA sequence of the present invention is depicted in SEQ ID NO. 4.
The present invention also relates to the use of the polypeptides of the present invention as a pharmaceutical—and a pharmaceutical composition/medicament—suitable for treating inflammatory skin conditions preferably comprising the polypeptide(s) of the present invention. For therapeutic purposes the polypeptide(s) of the present invention may be administered by any conventional means, either as an individual therapeutic agent or in combination with other therapeutic agents. The pharmaceutical compositions of the present invention can be adapted, using methods well known to those skilled in the pharmaceutical art, depending on the exact route of administration desired. Compositions of the present invention include, but are not limited to, those suitable for application to the skin via, for example, topical application and subcutaneous application. For the treatment of psoriasis, topical application is sufficient to give a therapeutic effect.
The present invention further relates to methods of producing the polypeptide of the present invention. Such methods would include recombinant expression of said polypeptide and in particular transforming an organism with a vector comprising a DNA sequence encoding the polypeptide, wherein said vector is capable of expressing said DNA sequence in said organism and growing said organism in conditions which allow the expression of said DNA sequence to produce said polypeptide. By growing it is meant increasing biomass, for example where the organism is a unicellular organism growing means increasing cell number. The term “organism” includes any organism that is suitable for the recombinant expression of the polypeptides of the present invention. Suitable recombinant expression systems include, but are not limited to, mammalian cell cultures, yeast and bacteria. Particularly preferred is E.coli. Vectors suitable for expression in host cell such as these would be readily apparent to the skilled man and include, for example vectors that harbour the T7 promoter, such as pET vectors, for expression in E. coli and other vectors suitable for expression in the yeast Pichia pastoris. The method of producing the polypeptide may also include the purification of the polypeptide. By purification it is meant obtaining the recombinant polypeptide from the production materials. Methods such as these could be employed during the Good Manufacturing Practice (GMP) production of these polypeptides.
The present invention further relates to a method of ameliorating an inflammatory skin condition comprising applying to a skin surface an effective amount of a composition comprising a molecule selected from the group consisting of:
The term “inflammatory skin condition” includes, for example, a human inflammatory skin condition and an animal inflammatory skin condition. In a preferred embodiment the inflammatory skin condition is selected from the group consisting of psoriasis, lichen planus, atopic eczema, irritant or allergic contact dermatitis, contact urticaria, infantile eczema and acne. The methods of the present invention are also useful in assisting wound healing, and in the treatment of burns, especially sunburn. Psoriasis is a chronic inflammatory skin condition characterised by the appearance of discrete psoriatic plaques. Psoriasis is associated with a number of changes in skin morphology. There is increasing evidence that pro-inflammatory cytokines play important roles in the pathogenesis of psoriasis. Current treatments include local topical administration of anti-inflammatory agents—typically a corticosteroid. Such treatments are not fully effective and are associated with unwanted side effects. Another therapeutic strategy is disruption of TNF-α function, but this also has been found to cause adverse reactions. There is a need therefore to provide further molecules that are effective in the treatment of inflammatory skin disorders, but which exhibit little or no adverse side effects and which ideally can be delivered by a non-invasive method, for example by application directly onto the inflammation.
Preferred for use in the method of the present invention is a molecule capable of inhibiting production and/or activity of IL-1α and/or IL-1β and/or capable of stimulating or enhancing the production and/or activity of IL-10.
TRX is a small (10-14 kDa), ubiquitous protein that is an important component of the cellular redox regulation system. Suitable TRX for use in the method of the present invention include TRX from (1) a prokaryote (e.g E. coli—SEQ ID NO. 7), (2) a plant (e.g Arabidopsis—SEQ ID NO. 8) and (3) an animal (e.g human—SEQ ID NO. 1). TRX can exist in a reduced state (wherein the two cysteines at the active site (Cys1-Gly-Pro-Cys2) provide a dithiol) and an oxidised state (wherein there is a disulphide bridge formed between the two cysteines at the active site). Under physiological conditions both redox states can exist—and both forms can be utilised in respect of the present invention. Furthermore, it is known that certain thioredoxins can exist in multimeric forms. For example, it is known that human TRX (hTRX) can form dimers wherein a disulphide bridge exists between Cys-73. These multimeric forms of the molecules may also be utilised, in addition to the monomeric form, within the methods of the present invention. It is however, preferred, that the molecule, for example thioredoxin, is in a substantially reduced state. By substantially reduced it is meant that >80%, preferably >90%, more preferably >95% of the molecules present are in a reduced state. A preferred molecule for use in the method is the recombinant human thioredoxin depicted in SEQ ID NO. 1—since this protein is an endogenous human protein, and is therefore unlikely to cause either adverse effects, or an immune response when administered to patients. Other molecules suitable for use in the method of the present invention include a protein that comprises a thioredoxin active site in which one or both of the cysteines at the active site are replaced by an amino acid other than cysteine. Examples wherein one or other of the cysteines is replaced include Cys1-Gly-Pro-Ala and Ala-Gly-Pro-Cys2. Surprisingly, it has been discovered that redox-inactive TRX molecules, in which both Cys1 and Cys2 are replaced, can also be successfully used in the present inventive methods. Furthermore, it has been shown that where the TRX molecule comprises additional cysteines other than at the active site these additional cysteines can also be replaced without any loss in activity. For example, in respect of human thioredoxin, which contains five cysteines (C32, C35, C62, C69 and C73) it has been shown that modified human thioredoxins comprising (1) C73A, (2) C32A, C35A and C73A; and (3) C32A, C35A, C62A, C69A and C73A retain biological activity. It has also been shown that activity is retained if cysteines present in the unmodified thioredoxin other than the active site cysteines are substituted and/or deleted. For example, the protein depicted in SEQ ID NO. 11 (C73A) has been shown to be active. It has also been found that the active molecules can be rendered inactive by heat treatment at 95° C. for 30 min, or 56° C. for 30 min, indicating that there is a structural feature associated with these molecules that is responsible for the observed activity. Thus the present invention further relates a molecule which comprises a region of three dimensional homology to a region present within the three dimensional structure of the active molecules disclosed in the present application, for example SEQ ID NO. 1, that are capable of ameliorating an inflammatory skin condition. Particularly preferred for use in the method are the polypeptides of the present invention, including the polypeptide sequences depicted in SEQ ID NO. 3, SEQ ID NO. 9 SEQ ID NO. 10 and SEQ ID NO. 11.
It has been shown that the molecule(s) described can be used to treat inflammatory skin conditions at extremely low application rates. Accordingly, the present invention further provides a pharmaceutical composition wherein the concentration of the active molecule within the pharmaceutical composition is preferably from 0.0001 to 0.5 w/v (1 μg/ml to 5 mg/ml) more preferably 0.0001 to 0.1% w/v, more preferably 0.0001% to 0.01% w/v, and still more preferably 0.0001% to 0.001% w/v. If the composition is a cream then it is particularly preferred that the active molecule is present at a concentration from 0.0001% to 0.02% w/v. Compositions comprising recombinant human thioredoxin in a substantially reduced, monomeric state are particularly preferred.
The application rate of the molecules described above to the skin surface is preferably 0.05 to 10 μg /cm2, more preferably 0.05 to 5 μg/cm2, and more preferably 0.1 to 1 μg/cm2. It is preferred that human thioredoxin in a substantially reduced, monomeric state is applied to the skin surface.
The present invention further relates to a method of treating inflammatory skin conditions comprising applying to a skin surface an effective amount of a composition comprising a molecule described above and an additional active ingredient. By additional active it is meant an ingredient that also has a pharmaceutical effect—which could be either additive or synergistic to the said molecule. Examples of additional active ingredients include lactoferrin (e.g. as depicted in SEQ ID NO. 5) and/or corticosteroids. The present invention also relates to a pharmaceutical composition comprising a molecule described above and an additional active ingredient. Preferred additional active ingredients include lactoferrin (e.g. as depicted in SEQ ID NO. 5), and/or corticosteroids and/or other topical medicaments suitable for the treatment of inflammatory skin conditions. A preferred composition is wherein the molecule is human thioredoxin depicted in SEQ ID NO. 1 and/or the modified TRX depicted in SEQ ID NO. 3 and wherein the additional active ingredient is lactoferrin, depicted in SEQ ID NO. 5. The compositions of the present invention may also comprise further ingredients, for example anti-oxidants such as glutathione, vitamin A, vitamin C, vitamin E, or indeed extracts from plants such as, for example, Aloe vera. The pharmaceutical compositions of the present invention can also be used in a combination therapy for the treatment of severe inflammatory skin conditions.
It is preferred that composition of the present invention is suitable for application to the skin. Accordingly the composition will typically be formulated as a solution, gel, lotion, ointment, cream, suspension, paste, liniment, powder, tincture, aerosol, transdermal drug delivery system, or similar in a pharmaceutically acceptable form by methods well known in the art. Substances that enhance the penetration of the active ingredients through the skin may also be added including, for example, dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone, alcohol, acetone, propylene glycol and polyethylene glycol. The compositions may be applied directly to the skin or via various transdermal drug delivery systems, such as patches.
The present invention further relates to the use of a polypeptide capable of ameliorating an inflammatory skin condition wherein said polypeptide is a modified thioredoxin, the modification comprising:
The present invention further relates to the use of the polypeptide depicted in SEQ ID NO.s 11 and 17 as a pharmaceutical.
The present invention further relates to the use of a polypeptide capable of ameliorating an inflammatory skin condition wherein said polypeptide is a modified thioredoxin, the modification comprising:
The present invention further relates to the use of the polypeptide depicted in SEQ ID NO.s 11 and 17 in the manufacture of a medicament suitable for application to a skin surface for ameliorating an inflammatory skin condition.
All sequences are provided herewith with an N-terminal methionine. For the avoidance of doubt, it should be understood that the present invention also includes sequences wherein the N-terminal methionine is absent.
The terms “TRX” and “Thio” are both used interchangeably as abbreviations for thioredoxin.
Mouse studies
Mice
Young adult (6- to 8-week old) male BALB/c strain mice obtained from the Specific Pathogen Free Breeding Unit (Alderley Park, Cheshire, UK) were used throughout these investigations.
Recombinant native human TRX (hTRX—SEQ ID NO. 1) or modified human TRX (SEQ ID NO. 3) were diluted to 16.7 μg/ml in aqueous cream BP and 30 μl (0.5 μg TRX) applied topically to the dorsum of both ears 2 hours prior to exposure at the same site to chemical or cytokine. Control mice received an equivalent volume of cream alone. In some experiments, animals received 0.5, 0.1 and 0.05 μg of TRX.
Chemicals and Exposure
The skin sensitising chemical 4-ethoxy-2-phenyloxazol-5-one (oxazolone; Sigma Chemical Co., St Louis, Mo.) was dissolved in 4:1 acetone:olive oil (AOO). Groups of mice received 25 μl of 0.5% oxazolone, or vehicle (AOO) alone, on the dorsum of both ears. Other control animals were untreated (naïve).
Recombinant murine TNF-α (specific activity 2×108 U/mg by L929 cytotoxicity assay; endotoxin level: 0.009 ng/μg) was obtained from Genzyme (West Malling, Kent, UK). Recombinant murine IL-10 (specific activity 1-2×108 U/mg; endotoxin level: <0.1 ng/μg) was purchased from R&D Systems (Oxon, UK). Cytokines were either supplied as, or reconstituted in, sterile solutions of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) as carrier protein. Cytokines were diluted with sterile PBS containing 0.1% BSA and were administered using 1 ml syringes with 30-gauge stainless steel needles. Mice received 30 μl intradermal injections into both ear pinnae.
Preparation and Analysis of Epidermal Sheets
Ears were removed either 4 h following exposure to chemical or IL-1β, or 30 min after treatment with TNF-α. Samples were split with the aid of forceps into dorsal and ventral ear halves. The dorsal halves were incubated for 90 min at 37° C. with 0.02M ethylenediamine tetra-acetic acid (EDTA; Sigma) dissolved in PBS. The epidermis was separated from the dermis using forceps and washed in PBS. Epidermal sheets were fixed in acetone for 20 min at −20° C. Following fixation, sheets were washed in PBS and then incubated at room temperature for 30 min with anti-mouse MHC (I-Ad/I-Ed) monoclonal antibody diluted to 5 μg/ml in 0.1% BSA/PBS. Sheets were then washed prior to incubation for a further 30 min with FITC-conjugated F(ab)2 goat anti-rat IgG, diluted 1:100 in 0.1% BSA/PBS. Finally, sheets were washed in PBS and mounted on microscope slides in Citifluor (Citifluor Ltd., London, UK) and sealed with nail varnish. Samples were examined in a blinded fashion by fluorescence microscopy and the frequency of stained cells assessed using an eyepiece with a calibrated grid (0.32×0.213 at ×40 magnification). For each sample 10 consecutive fields in the central portion of the ear were examined.
Measurement of Epidermal Cytokine Production
Ears were removed 2 h following exposure to 0.5% oxazolone and prepared for explant culture under aseptic conditions. Ears were washed immediately in 70% ethanol, rinsed in PBS and were split with the aid of forceps into dorsal and ventral halves. Dorsal halves were floated on 250 μl RPMI-1640 medium in 24-well tissue culture plates (1 dorsal ear half/well). Supernatants were collected after 16 h of culture, pooled for each mouse and centrifuged at 150 g for 5 min prior to storage at −70° C. The IL-10 content was measured in supernatants using the Bio-Plex™ cytokine array system according to manufacturer's instructions (Bio-Rad Laboratories, Hercules, Calif., USA).
Preparation and Analysis of Dendritic Cells (DC)
Draining auricular lymph nodes were excised 18 h following treatment with chemical, or 4 h and 17 h following administration of the cytokines TNF-α and IL-1β, respectively. Nodes were pooled for each experimental group. A single cell suspension of lymph node cells (LNC) was prepared under aseptic conditions by mechanical disaggregation through sterile 200-mesh stainless steel gauze and resuspended in RPMI-1640 growth medium (Gibco, Renfrewshire, UK) supplemented with 25 mM HEPES, 400 μg/ml streptomycin, 400 μg/ml ampicillin and 10% heat-inactivated fetal calf serum (RPMI-FCS). Viable cell counts were performed by exclusion of trypan blue dye and the total cellularity per lymph node recorded. The cell concentration was adjusted to 5×106 cells/ml in RPMI-FCS and DC-enriched populations were prepared by discontinuous gradient centrifugation on Metrizamide (Sigma Chemical Co.; 14.5% in RPMI-FCS). The frequency of DC in such low buoyant density fractions was assessed routinely by direct morphological examination using phase contrast microscopy.
TRX in aqueous cream (0.5 μg in 50 μl) was applied topically to two skin sites (each 2 cm2 area) identified on non-sun-exposed buttock or hip. A further two sites on the contralateral buttock or hip received 50 μl of aqueous cream alone. Two hours later, volunteers received two 50 μl intradermal injections of 500 U of homologous recombinant TNF-α diluted in sterile normal saline and two control injections of 50 μl of sterile saline alone to paired sites (one exposed previously to TRX and one exposed to cream alone). Punch biopsies (6 mm) were taken under local anaesthesia (1% lignocaine) from each of the treated sites 2 h later.
Epidermal Langerhans cells (LC) were identified on the basis of their expression of CD1a; a membrane determinant that characterises LC in human epidermis. To stain for LC, biopsies were placed immediately in 0.02M ethylenediamine tetraacetic acid (Sigma, St Louis, Mo., USA) dissolved in phosphate buffered saline (PBS) and incubated for 2 heat 37° C. The epidermis was separated from the dermis using forceps, washed in PBS and fixed in acetone at −20° C. After washing in PBS, epidermal sheets were incubated at room temperature for 30 min with monoclonal antibodies specific for CD1a [clone NA1/34 (mouse IgG2a); DAKO Ltd, Cambridge, UK] diluted to 10 μg/ml in PBS containing 0.1% bovine serum albumin (BSA). Sheets were washed prior to incubation for a further 30 min with fluorescein isothiocyanate-conjugated goat F(ab′)2 anti-mouse immunoglobulins (DAKO) diluted 1:100 in 0.1% BSA/PBS. Finally, sheets were washed in PBS and mounted on microscope slides in Citifluor (Citifluor Ltd., London, UK) and sealed with nail varnish. The identity of each slide was then masked using tape.
Samples were examined by fluorescence microscopy and the frequency of stained cells assessed in a blinded fashion using an eyepiece with a calibrated grid (0.32×0.213 mm at ×40 magnification). For each sample, 50 consecutive fields were examined. The identity of each slide was revealed after all samples have been counted. Results are expressed as the mean±SD number of cells/mm2.
The purpose this experiment was to determine whether topical application of native hTRX to mouse skin was able to influence the integrity of LC migration induced by subsequent exposure at the same site to oxazolone, a potent contact allergen. The results of a representative experiment are illustrated in
Previous studies have provided clear evidence that the migration of epidermal LC, in both mouse and man, is dependent upon the availability of certain cytokines and chemokines, two of those known to be of particular importance being interleukin- 1β (IL-1β) and tumour necrosis factor α (TNF-α). There is a precedent for perturbation of cytokine function resulting in compromised LC migration. In the next experiments we therefore investigated whether hTRX could affect LC migration induced by either IL-1β or TNF-α. The results of a representative experiment are displayed in
In a parallel series of experiments the same question as addressed in Experiment 2 was explored, but using a supplementary endpoint. In this case the endpoint used was the accumulation of dendritic cells (DC) in skin-draining regional lymph nodes. The relevance of this measurement is that the epidermal LC that are provoked to migrate from the skin traffic via afferent lymphatics to draining lymph nodes (in order to interact with the adaptive immune system). The effectiveness of LC mobilisation can therefore be measured either as a function of the loss of LC from the epidermis, or as a function of their subsequent accumulation in skin-draining lymph nodes. A representative experiment is illustrated in
Most biological properties of TRX are considered to be a function of the redox activity of this protein. There are available redox-inactive mutant variants of the protein that have discrete amino substitutions that render the protein redox-inactive. One such mutant is C32A/C35A, as depicted in SEQ ID NO. 3. In another series of experiments the ability of C32A/C35A to inhibit LC migration was investigated and compared with the activity of native hTRX (SEQ ID NO. 1). A representative experiment is shown in
In subsequent experiments the relative potency of native hTRX (SEQ ID NO. 1) and of C32A/C35A (SEQ ID NO. 3) were compared with respect to inhibition of LC migration. In one experimental design various concentrations of the redox-inactive mutant protein were compared with a single concentration of the native hTRX. The results of a representative experiment are summarised in
In parallel investigations the same experimental design was employed with the reverse orientation. That is, a dose response was performed with the native hTRX and the results compared with the effects of a single dose of the redox-inactive mutant. A representative experiment is illustrated in
In the next series of experiments the impact of hTRX on the integrity of LC migration in humans was investigated using healthy adult volunteers. The results obtained using two such volunteers are illustrated in
This experiment was designed to show that the C32A/C35A modified human TRX was redox inactive. The assay was run at room temperature for 15 min and the reduction of dithionitrobenzoic acid (DTNB) followed at 412 nm overtime with a spectrophotometer. The reaction mixture contains an excessive concentration of NADPH that is consumed by the TRX reductase to reduce TRX. After that, TRX reduces preferentially DTNB and TRX is recycled in its reduced form by the reductase and NADPH.
In a separate series of experiments the influence in mice of topical treatment with TRX on the elaboration by skin cells of IL-10 was measured. A representative experiment is shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 0413114.0 | Jun 2004 | GB | national |
| 0504426.8 | Mar 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2005/002300 | 6/10/2005 | WO | 00 | 9/25/2008 |
